STUDIES ON RESTRICTION ENDONUCLEASES IN ACETIC-ACID BACTERIA AND ALLIED ORGANISMS .7. PURIFICATION OF RESTRICTION ENDONUCLEASE FROM ACETOBACTER-PASTEURIANUS IFO 13752 (APAORI) AND ITS PROPERTIES

A restriction endonuclease, designated ApaORI, was purified from cell-free extracts of A. pasteurianus IFO 13752 by streptomycin treatment, ammonium sulfate fractionation, phosphocellulose treatment, hydroxyl-apatite and heparin-Sepharose CL-6B column chromatography, and gel filtration using a Superose 12 (HR10/30) column. The purified enzyme was homogeneous on polyacrylamide gel disc electrophoresis. The molecular weight of the purified enzyme was found by gel filtration to be 21,000 daltons. The isoelectric point of the purified enzyme was neutral (6.9). The purified enzyme cleaved λ, ϕX174 RF I, M13mp7 RF I, and pBR322 DNAs at 18 or more, 2,4 or more, and 4 or more sites, respectively. The purified enzyme worked best at 37°C and pH 7.5 in a reaction mixture (50μl) containing 1.0 μg λDNA, 10 mM Tris-HCl, 7 mM 2-mercaptoethanol, 7mM MgCl2, and 50 mM NaCl. However, the purified enzyme did not require NaCl for its reaction. The purified enzyme recognizes the palindromic pentanucleotides 5'-CC (AorT)GG-3' and cuts between C and A (or T), producing 5'-cohesive mononucleotide extensions (isoschizomer of BstNI). © 1990, Applied Microbiology, Molecular and Cellular Biosciences Research Foundation. All rights reserved.